Isolation of penicillin



Patented Apr. 3, 1951 2,547,640 l-C E 2,547,640 ISOLATION OF YPENIGILLI'N Leon Goldman, Ny'ack; N.

canCyanamid Company, corporation of Maine Y., assi hor to Amati- New'York, N. Y., a

No Drawing. Application-March 17,1948,

Serial No. 15,490

17 Claims. (Cl. 260*302) This invention relates to penicillin and the pro= duction and purification thereof. More particu larly; the invention relates-to an improvement in processes o'ipurifying penicillin and in separating penicillin G from other penicillins and impurities associated therewith.

. The discovery of penicillin and its remarkable therapeutic value has ledto its production on alarge commercial scale; Early processes yielded a relatively impure product of variable composiuse containing uhkonwh substances in addition to the desired antibiotic.- Investigations concern= irig the nature-6f the product led to the-discovery tnat commertiauy produced penicillin contained several active peni'cil-lins which were variously designated penieillin F,- periiillin K, penicillin X,- penicillin and the like Of these, penicillin G, also called benzylpenicillin, is considered the most important.

unrert'unateiy, the presently known methods or extracting penicillin from the fermentation liquor have not been satisfactory because of the many process steps involved and the great loss of penicillinsuffer'ed while isolating the pure mate: rial; Although pure penicillin and, in p c lar, crystalline" salts of penicillin G have been ob-' tained, it hasbeen considered necessary to first prepare a relatively concentrated solutionof penicillin in an anhydrous organic solvent and precipitate the penicillin therefrom as an insoluble salt. It has also been considered necessary to conduct the crystallization operation at very low temperatures to avoiddecomposition of the penicillin; The yields have been low. All or the factors make known crystallization rocedures expensive. Also, some previously usedprocesses were not capable of separating penicillin G from other less desirable penicilins. 'I have discovered that relatively pure penicillin in salt forin can be obtained ingoo'd yields by" asirfiple process from a ueous solutions of Deni cillin at substantially neutral conditions and at room temperature. This discovery has been utilized-011a large scale to recover penicillin from its aqueous fermentation liquor andhas greatly simplified the problem of obtaining pure crystalline penicillin G.

In accordance with my new process an aqueous 's oiution 0f li hidillil'i is prepared: solutions'of 5,000 or more Oxford units of penicillin per mi.-

are preferred, but it is not necessary for the operation-of the process that the aqueous solution be so strong. Solutions having 5,000 or more Qaford units-perml. may be obtained by concentrating filtered fermentation liquor, containing the usual'commer'cially' achieved quantities of penicillin" by vacuumevaporation- .Or otherwise, ingasolution of penicillin-mawateriminisc'ibl organie solvent'with an aqueous so'lu tionwcon ing an alkaline substance and/or bulier-a ts 5. t he final aqueous extract containing the peni=- "process. 1 zafnines 'amino n-octane, alnha-phenylethylamine, cyclocill'iii sheuld be adjusted to hav a' pH within the" range of about pH 4.5 to 8.5 by treatment if the solution with an alkali- (irfacid and/or tuner agents Under these eoriditions the penicillin iiithe aqueous solution is most probably in'the forth of one of its salts, disassoc'iated to some extent,

as would be xpected. various salting out agents are also" added. These include ammonium sulfate, ammonium chloride, sodium Chloride,

sodium sulfate, magnesi m sulfate, lithium sul fate, calciuiiinitiate, sodium tetraphosphate and various alkali metal phosphates; py ophosphate's",

mtaphos'phat's aiidth like. SaIting dlit agents;

are Well kliiiWh to those Skilled in the art, alfid the se'ection and use thereof is susceptible t6 considerate variation. The V out ag fit in the Solution may vary considerably and will be fiom' about 5% to 40% by Weight,

b a'sid on the total weight of the aqueous solution. 1

To the solution or penicillin salt described above there is thei'i added an M a San with penicillin having a favorable eaten n corficint for extraction into a water immiscible,

solvent from the aque us soiu'tion under the anions" desribed. The amifie isadded as t e free base or as one of its acid salts. v When the free amihe' is: added, an acid should be added at slilfistailtiallythe same time so that the pH of the aqueous solution is maintained Within the range pH 4.5 to pH 8.5.

An apparent distinction eiiists between suitability or the tertiary amines on the one hand seeiridafy' amines that are suitable for use" in the.

process of the present invention appear to have a relatively narrow range of basicity and ingenera-l, may be weaker than the tertiary amines. As will be apparent from the numerous examples which follow, primary and secondary amines having an ionization constant of between about 1 10- and 1X10"- are suitable for use in the Among such primary and secondary may be mentioned isoamylamine, 2-

lieiiyl'amine, butylamine, ethylamine, isobutylamine, methylamine, diisopropylamine, diethylamine, diisoamylamine, diisobutylamine, dimethylamine, piperidine, and still other primary andsecondary amines; v Q-bviously, some of these amines are better than-others. in the process, and different aminesgive better results with particular solvents.- v Optimum conditions can readily be determined by those skilled in the art fol lowing the teachingsoi the presentinvention.

The amine or amine salt'is added fin at least amine which for SI stoichiometric proportions to the penicillin present in the aqueous solution but more preferably in a large excess within the range of from f penicillin having a potency of 6,430 unitS/mL were added 150 grams of ammonium sulfate, 4.6

1 to 20 times or even more, as much of the amine EXAMPLE 1 To one liter of a solution of crude sodium salt as penicillin on a molecular basis. The solu- 5 ml. of 85 hos horic acid, and 30.8 ml. of tritions may be maintained dur ngthis time at room I ethylamng p resulting solution of pH 7.4.? m m although lower. temperatures qr was extracted with four 100 ml portions of cold slightly higher temperatures will not detract serichloroform The four extracts assayed respec us1y mm the Process tively 43,000 units/ml., 14,700 units/ml, 4,500 To the aqueous solution, as ust described, is un ts /m1 and 2 200 units/ml totaling 100% of then added a water immiscible organic solvent ori 'i unitg in which the substituted ammonium penicillin The gfirst three; extracts were combined and salt. formed as a result of-addition of the amine, is. soluble. Chloroform appears tohe the best 2232 3552 g zg ig i gg fig ig fifi g g gfi water immiscible solvent for the triethylammonil5 acetate wa eva oiflation was con'tinued to, umsalt r penicillin and is preferred. Other waremove i chlrofoffm and finally 5 m1 ter. immiscible organic solvents such as ethylene acetone was added Aitel: chilling overnight 22 31: s: fjggggjf fgff ggt g g gfi ggi crystals were filtered and washed with acetone, fi etone may a be usedho'wever yielding 3.13 g. of crystalline triethylammonium After intimately contacting the liquids and gg g g clilln, alslsaymg 1,2kflt5 umts/mg. and separating them by decantation or otherwise, the Y P 0 in by Weig water immiscible organic solvent containing dis- EXAMPLE 2 v solved penicillin is removed from the aqueous L To 149 liters of a solution of crude sodium salt phase. The extraction process may be conof penicillin having a potency of 6490 units/1111' ducted in separatory funnels,.in counter current were added 23 kg of ammonium sulfate 570 m1 cmmms in cPminuous centrifugal SeParatm's of 85% phosphoric acid and 5.1 liters of triethyl typias known m W The of amine. The resulting solution of pH 7.4 was examine salt of penicillin which contains the salt of acted four times with equal volumes of ch1or0 pemmnmgnas well as cwespondmg Salts 0f form. The total chloroform extract (33.35 liters), other penicillins, may then be evaporated at room containing 936 million units (96 4% of the orig? temperature, more or less, invacuo, if desired, and Hal units) was 'concentrated a Vacuum still, tn? penicmins-alt mom-16.1 9 therefrom Under until crystals formed One liter of amyl acetate sultable condlllons pemclmn m be recovered was added and distillationwa continued to retherefron-l by slmple crystamzatmn" move more chloroform. Then 2 liters of acetone pal-'mculgfly advantageous way of obtaming was added and the slurry was agitated and gg fl fi 3 i gzgfig zg sfifi g zfifi 33 2 1 chilled overnight. The resulting crystals were a'myl acetate in which i desired periicimn G filtered, washed with acetone, and dried in vacuo. amine salt is less soluble than the corresponding 40 gg g fig gfi g ii gf gggggfifi g ggs g 3 5: amine salts of penicillin K and other less desirunits/ able forms of penicillin. The concentration is EXAMPLE 3 continued until the first solvent has been re- I moved. Evaporation is then stopped, and the so- To 500 ml. of an aqueous solution of 6.5 million lution is cooled and allowed to stand until no more units of sodium penicillin, 0.218 mole of triethyl-; crystals of the desired amine salt of penicillin G ammonium chloride and 65 grams of ammonium separate from solution. The product may be sulfate, was added 0.4-0.5 ml. of 7.5% sodium washed with acetone, and the highly purified hydroxide to adjust the pH to 6.5. The resulting crystalline salt ofpenicillin G obtained. solution was extracted with six ml. portions of Another method of obtaining the pure crystals 5 ethylene dichloride, and the extracts were como f penicillin G is to evaporate off the original orbined and assayed. L ganic solvent used in the extraction until penicil- The above procedure was repeated using the lin Gprecipitates from solution. The crystalline following solvents: methyl n-propyl ketone, material may then be washed with methyl ethyl methyl isobutyl ketone, n-butanol, sec-butanol, ketone or other solvent in which the penicillin and n-pentanol.

' Table I Number of Total T t P C Solvent ii it n o f Po-tency' Urgtsin erpf-en (mm of Extract units/ml. Extract Orig nal each X10 Units Ethylene dichloride 6: 50 293 5, 225 1.53 23. 6 Methyl n-propyl kotone. 6:50 286 4,600 1.32 20.3 Methyl isobutyl ketone. 6'50 292 2,050 0.598 9.2 n-butanol; M0 295 14,000 4.13 03.0 scobutanol 1=40,0;50 358 15.000 5.37 82.7 n-pcntanol 6:50 275 12,600 3.47 53.3

G is relatively insoluble, whereby the impurities EXAMPLE 4 are removed from'the crystalline material.

- To illustrate the'invention in greater detail, the following examples are ofiered. It should be understood that these are merely illustrative and should not be construed; to liinitjthe invention to the particular detailsfthereof. N All parts are by weight unless otherwise indicated. e

A solution of cyclohexylammonium phosphate, prepared by mixing together 21.9 grams of cycle hexylamine, 5.5 m1. of phosphoric acid and ml. of water, was added to a solution of 5.0 grams of sodium penicillin (6.5 million units) and 65'gram's of ammonium sulfate in' 100 mL-of 1T0 :theresulting solution 110'0 :mlrorwater' i wasaddedgand 4.5 mror 0.5 M sodium'hydroxide to adjust the. pI-I'to 7.39. six .40 ml.;chloroform' extractions weremade, and'assay of the combined chloroform extract was 9,460 units/ml. (38% :of the original units). The chloroform extract'was dried over anhydrous sodium sulfate and distilled todryness-in vacuo at room temperature; yielding 23 grams of cyclohexylammonium penicillin $3.8! saying BI'ZLHIIitS/mg.

EXAMPLE 5 solution of :1 g. of sodium penicillin (assaying-l-300 units/mg), grams of ammonium sulfate; "53ml; of cyclohexylamine, and 4.0 ml. of 85% phosphoric acid in 200 ml. of "water was adjuste'cl to pH'5.39by adding 0.5 M NaOI-I, and ex 'tracted. With'siX' 20 ml. portions of chloroform.

Th'eiizcombined chloroform extract, which assayed 4,800 units/ml:,--was chilled onehour, :and the crystals of cyclohexylammonium penicillin were removed by filtration and washed twice with mlrportions of petroleum ether. The yield was 0.36 g. assaying 1,318 units/mg.

EXAIWPLE' 6 Six 1.00 gram samples of sodium penicillin assaying 1,530 units/mg, and 20 gram portions of ammonium sulfate were dissolved in 50 ml. of Water, respectively. To each was added,:respectively. a solution of 0.0514 mole of an amine selected from the listin Table II and 4.33 ml..of hydrochloric "acid, sp. gr. l.19,-in 10 ml. of water and-the pHad'justed withl.5 2 m1. of 75% NaOH to pH 7.50. The resultingzsolutions were adjust-- ed to "pH 6.50. and water was added to "bring the volumes to '100; ml. The solutions were extracted with .six 10 ml. portions of chloroform, andlthe combined chloroform extracts were assayed for T0150 'ml. of an aqueous solution of 6.5'million units of sodium penicillin and '65 grams of ammonium sulfate was added 1'75 ml. of a solution of 12.5 grams of N, N'-dimethylpiperazine and 40ml. of 85% phosphoric acid. The resulting solution'was diluted to 325 ml., 1.2 ml. of'phosphori-cyacid was added to bring the pH to 6.50, and then the solution was extracted withsix 40 mliportions of chloroform. The combined chloroform extract, 232 ml. assaying 23,950 units/ml, was dried over'anhydrous sodium sulfate-and distilled in vacuo to dryness, yielding 5.4 gramsof "N, =.N-dimethylpiperazinium penicillin, assaying 1,050 units/mg.

I I EXAMPLE' 3 I A solution of 6.5.m1'llion units of sodium peniicillin and 6.5 grams of ammonium sulfate in .150

ml. of water was combined with a solution of 22.1 grams of N-ethylpiperidine and .6.5 m1; of

85 phosphoric acid in .150 ml. of water. The re uiting solutionawasadiusted to pIl 31.40 by.add-.

6 1113314 ml;::o;f 0.5 M then extracted-withrsix 40 ml. portionszof .chloro-r form. The combined ohloroformextract was: dried over anhydrous sodium sulfate and distilled. invacuo to dryness. The yieldof N-ethylpiperidinium penicillin was 4.4 grams, assaying.. l ;22.6

unitS/ EXAMPLE 9 A solutionof 61.2 million units of sodium penicillin, .520 grams of ammonium sulfate, andfl'] ml. of 2.18 .M triethylammonium chloride solu:

tion was diluted with water to 3,080 ml. and divided into eight portions of 385 ml. each. To each was added, respectively, the following quantities of 2.18'M'triethylammonium chloride: 1.9.4.1, 69.1, 44.1, 34.1, 24.1, 14.1, 4.1,.and0ml... and each was diluted. to 500. ml. and the pHadJ'usted to 6.08- 618. Thelsolutions Were extracted with v.six ,50 m'l., portions ofchloroform, and the pooled chloro form extracts'were evaporated in vacuo to dryness. Theresidues were crystallized from chloroform-ethyl acetate mixtures to yield pure triethylammonium penicillin in the amounts shown, 1n Table Ill Table HI 'Tricthylammonium Penicillin Molar Excess Unit Per cthylamine Cent Yield.

units/ weight,

g. mg.

coo: :ncnwmd: 000006 EXAMPLE 1o Tosolutions of 6.5 million units of sodium penicillin in ml. of 2.18 M triethylammonium ,chlo ride solution, each of the salts in Table IV was added, respectively, each was diluted with water to 325 ml., and the pH of each was adjusted to 6.00 by adding 7.5% sodium hydroxide or 18%,hydrochloric acid as needed. The resulting solutions were extracted with. six 40 m1. portions of chloroform, the combined chloroform extracts were dried over anhydrous sodium sulfate, and the extracts were distilled to dryness in vacuo. The residues were then crystallized from chloroform-ethyl acetate to yield pure .triethylammonium penicillin.

.Table IV.

Triethylammonium Combined CHCls Extract Penicillin Salts Added g ml. units/n11. Grams Units/mg.

.I claim:

' 1. .A processof obtaining substituted ammo, nium salts of penicillin from aqueoussolutions thereof :which comprises the steps of preparing an aqueous solution of penicillin having a-pH within the range 4.5 to 8.5 and containing from about 53% to 40% by weight of ,a ;salting ..out

sodium hydroxide and-:was

agent and adding thereto a large stoichiometric excess, based on the penicillin present in said solution, of an organic amine having a dissociation constant of at least 1 l0 while maintaining the pI-I within the range 4.5 to 8.5, thereafter mixing the aqueous solution containing the resulting substituted ammonium penicillin salt with a water immiscible organic solvent, separating the organic solvent from the aqueous phase and recovering from said organic solvent the substituted ammonium salt of penicillin.

2. A process of obtaining substituted ammonium salts of penicillin from aqueous solutions thereof which comprises the steps of preparing an aqueous solution of penicillin having a pH within the range 4.5 to 8.5 and containing between about 5% to 40% by weight of a salting out agent and. adding thereto from about 1 to 20 mols, based on the penicillin present in said solution, of an organic amine having a dissociation constant of a least 1 l0- while maintaining the pH within the range 4.5 to 8.5 and thereafter mixing the aqueous solution with chloroform, separating the chloroform from the aqueous phase and recovering the substituted ammo-- nium salt of penicillin by evaporation of the chloroform.

3. A process of obtaining crystalline substituted ammonium salts of penicillin which comprises the steps of preparing an aqueous solution containing penicillin G having a pH within the range 4.5 to 8.5 and containing between about 5% to 40% by weight of a salting out agent and adding thereto from about 1 to 20 mols, based on the penicillin present in said solution, of an organic amine having a dissociation constant of at least l while maintaining the pH within the range 4.5 to 8.5, and thereafter mixing the aqueous solution with chloroform, separating the chloroform from the aqueous phase and evaporating the chloroform until part of the volume thereof has been reduced, adding amyl acetate, continuing the evaporation of the mixed solvents until the amine salt of penicillin G has separated therefrom, and thereafter separating the insoluble amine salt of penicillin G from its mother liquor.

4. A process of obtaining substituted ammonium salts of penicillin from aqueous solutions thereof which comprises the steps of preparing an aqueous solution of penicillin having a pH within the range 4.5 to 8.5 and containing from about 5% to 40% by weight of a salting out agent and adding thereto a large stoichiometric excess, based on the penicillin present in said solution, of a tertiary amine having a dissociation constant of at least 1 l0 while maintaining the pH within the range 4.5 to 8.5, thereafter mixing the aqueous solution containing the resulting substituted ammonium penicillin salt with a water immiscible organic solvent, separating the organic solvent from the aqueous phase and recovering byv evaporation of the organic solventthe substituted ammonium salt" of penicillin.

5. A process of obtaining substituted ammonium salts of penicillin from aqueous solutions thereof which comprises the steps of preparing an aqueous solution of penicillin having a pH within the range 4.5 to 8.5 and containing from about 5% to 40% by weight of a salting out agent and adding thereto a large stoichiometric excess, based on the penicillin present in said solution, o'f'a secondary amine having a dissociation con stant within the range 1 l0 to 1 l0- while maintaining the pH within the range 4.5 to 8.5,

thereafter mixing the aqueous solution containing the resulting substituted ammonium penicillin salt with a water immiscible organic sol-' vent, separating the organic solvent from the aqueous phase and recovering by evaporation of i the organic solvent the substituted ammonium salt of penicillin.

6. A process of obtaining substituted ammonium salts of penicillin from aqueous solutions" thereof which comprises the steps of preparing an aqueous solution of penicillin having a pH within the range 4.5 to 8.5 and containing from about 5% to 40% by weight of a salting out agent and adding thereto a large stoichiometric excess,

based on the penicillin present in said solution,

of a primary amine having a dissociation constant within the range l l0- to 1x10 while maintaining the pH within the range 4.5 to 8.5, thereafter mixing the aqueous solution contain-" ing the resulting substituted'ammonium peni-' cillin salt with a water immiscible organic solvent, separating the organic solvent from the aqueous phase and recovering by evaporation of" an aqueous solution of penicillin having a pH within the range 4.5 to 8.5 and containing be-f tween about 5% to 40% by weight of a salting out agent and adding thereto from about 1 to 20:

mols, based on the penicillin present in said solution, of a tertiary amine having a dissociation constant of at least 1 l'0 while maintain-' ing the pH within the range 4.5 to 8.5, and thereafter mixing the aqueous solution with chloroform, separating the chloroform from the aqueous phase and recovering from the chloroform the substituted ammonium salt of penicillin.

.8. A process of obtaining substituted ammonium salts of penicillin from aqueous solutions thereof which comprises the steps of preparing an aqueous solution of penicillin having a pH within the range 4.5 to 8.5 and containing between about 5% to 40% byweight of a salting out agent and adding thereto from about 1 to 20 mols, based on the penicillin present in saidsolution, of a secondary amine having a dissociation constant within the range l X 10- to ix 10- while maintaining the pI-I within the range 4.5 to 8.5, and thereafter mixing the aqueous solution with chloroform, separating the chloroform from the aqueous phase and recovering from the chloroform the substituted ammonium salt of penicillin.

9. A process of obtaining substituted ammonium salts of penicillin from aqueous solutions thereof which comprises the steps of preparing an aqueous solution of penicillin having a pH within the range 4.5 to 8.5 and containing be tween about 5% to 40% by weightrof a salting out agent and adding thereto from about 1 to 20. mols, based on the penicillin present in said solution, of a primary amine having a dissoeia tion constant within the range 1 X 10- to l x 10-' while maintaining the pH within the range 4.5 to 8.5, and thereafter mixing the aqueous solution with chloroform, separating the chloroform from the aqueous phase and recovering from the chloroform the substituted ammonium salt of penicillin.

10. A process of obtaining substituted ammonium salts of penicillin from aqueous solutions thereof which comprises the steps of preparing vmols, based on the an aqueous solution of penicillin within the range 4.5 to 8.5 and containing between about to 40% by weight of a salting out agent and adding thereto from about 1 to mols, based on the penicillin present in said solution, of an organic amine having a dissociation constant of at least 1 10- and thereafter mixing the aqueous solution with secondary butanol, separating the secondary butanol from the aqueous phase and recovering the substituted amhaving a pH monium salt of penicillin by evaporation of the butanol.

11. A process of obtaining substituted ammonium salts of penicillin from aqueous solutions thereof which comprises the steps of preparing an aqueous solution of penicillin having a pH within the range 4.5 to 8.5 and containing between about 5% to by weight of ammonium sulfate and adding thereto from about 1 to 20 lution, of an organic amine having a dissociation constant of at least 1 10- while maintaining the pH within the range 4.5 to 8.5, and thereafter mixing the aqueous solution with chloroform, separating the chloroform from the aqueous phase and recovering the substituted ammonium salt of penicillin by the evaporation of the chloroform.

12. In a process of recovering penicillin from aqueous solutions thereof the improvement which 4 comprises the steps of adding to an aqueous solution, containing at least 5,000 Oxford units of penicillin per milliliter and having a pH within the range 4.5 to 3.5 and containing between about 5% to 40% by weight of a salting out agent, at least a stoichiometric quantity of triethylamine, based on the penicillin cillin is separated therefrom.

13. In a process of recovering penicillin from aqueous solutions thereof the improvement which about 5% to 40% by weight of a salting out agent, at least a stoiohiometric quantity of beta-aminoon the penicillin present in said about 5% to 40% by weight of a salting out agent, at least a stoichiometric quantity of N,N'-diof the aqueous solution within the range 4.5 to 8.5,

penicillin present in said so- 10 and thereafter intimately mixing the aqueous solution with chloroform whereby the N,N'-dimethylpiperazinium salt of penicillin is extracted from the aqueous phase into the chloroform, separating the chloroform from the aqueous phase, and evaporating the chloroform until the N,N'- dimethylpiperazinium salt of penicillin is sepa rated therefrom.

15. In a process of recovering penicillin from aqueous solutions thereof the improvement which comp-rises the steps of adding to an aqueous solution, containing at least 5,000 Oxford units of penicillin per milliliter and having a pH within the range 4.5 to 8.5 and containing between about 5% to 40% by weight of a salting out agent, at

least a stoichiometric quantity of N-ethylpiperidine, based on the penicillin present in said solution, while maintaining the pH of the aqueous solution within the range 4.5 to 8.5, and thereafter intimately mixing the aqueous solution with chloroform whereby the N -ethylpiperidiniurn salt of penicillin is extracted from the aqueous phase into the chloroform, separating the chloroform from the aqueous phase, and evaporating the chloroform until the N-ethylpiperidinium salt of penicillin is separated therefrom.

16. A process of nium salts of thereof which comprises the steps of preparing within the range 4.5 to 8.5 and containing between about 5% to 40% by weight of sodium tetraphosp-hate and adding thereto from about 1 to 20 mole, based onthe penicillin present in said solution, of an organic amine having a dissociation constant of at least 1 10- while maintaining the pH within the range 4.5 to 8.5, and thereafter mixing the aqueous solution with chloroform, separating the chloroform from the aqueous phase and recovering from the chloroform the substituted ammonium salt of penicillin.

17. A proce s of obtaining crystalline substituted ammonium salts of penicillin G which comroform from the the chloroform until crystals of the substituted.

ammonium salt of penicillin G separate therefrom and washing said crystals with methyl ethyl ketone.

LEON GOLDMAN.

REFERENCES CITED The following references are of record in the file of this patent:

Heyden Report, CMR-H-II, May 22, 1944, pp. 1 and 2 (abstracted in Science, vol. 104, Nov. 8, 1946, pp. 433 and 450).

Merck Report, CMR-M-77, Sept. 28, 1945, pp. 1, 2 and 3.

British Report, CMR Br-14'1, CPS 382, Jan. 22, 1945, page 1.

British Report, CMR-Br-234, (PB 79927) CPS-687, Feb. 12, 1946, received in 'U. S. April '18, 1946, pp. 1 to 5. 

1. A PROCESS OF OBTAINING SUBSTITUTED AMMONIUM SALTS OF PENICULLIN FROM AQUEOUS SOLUTIONS THEREON WHICH COMPRISES THE STEPS OF PREPARING AN AQUEOUS SOLUTION OF PENICILLIN HAVING A PH WITHIN THE RANGE OF 4.5 TO 8.5 AND CONTAINING FROM ABOUT 5% TO 40% BY WEIGHT OF A SALTING OUT AGENT AND ADDING THERETO A LARGE STOICHIOMETRIC EXCESS, BASED ON THE PENICILLIN PRESENT IN SAID SOLUTION, OF AN ORGANIC AMINE HAVING A DISSOCIATION CONSTANT OF AT LEAST 1X10-5 WHILE MAINTAINING THE PH WITHIN THE RANGE 4.5 TO 8.5, THEREAFTER MIXING THE AQUEOUS SOLUTION CONTAINING THE RESULTING SUBSTITUTED AMMONIUM PENICILLIN SALT WITH A WATER IMMISCIBLE ORGANIC SOLVENT, SEPARATING THE ORGANIC SOLVENT FROM THE AQUEOUS PHASE AND RECOVERING FROM SAID ORGANIC SOLVENT THE SUBSTITUTED AMMONIUM SALT OF PENICILLIN. 